Abstract

Background

Saturated fatty acids have been shown to cause insulin resistance and low-grade chronic inflammation, whereas unsaturated fatty acids suppress inflammation via G-protein coupled receptor 120 (GPR120) in macrophages. However, the anti-inflammatory effects of unsaturated fatty acids in adipocytes have yet to be elucidated. Hence, the aims of the present study were to evaluate the anti-inflammatory effects of eicosapentaenoic acid (EPA) via GPR120 in adipocytes.

Methods

We used 250 μM palmitate as a representative saturated fatty acid. 3T3-L1 adipocytes were used for in vitro studies. We further evaluated the effect of EPA supplementation in a high-fat/high-sucrose (HFHS) diet-induced adipose tissue inflammatory mouse model.

Cell culture, treatment, and harvesting

3T3-L1 murine pre-adipocytes were purchased from American Type Tissue Culture Collection and differentiated according to a previously described standard protocol. [10] Fully differentiated 3T3-L1 adipocytes were exposed to 250 μM palmitate for 30 min, 60 min, or 24 h with or without a 6-h pretreatment with 50 μM EPA-Na. Cells were harvested by RIPA buffer with phosphatase and protease inhibitors (nacalai tesque) for protein extraction.

Animal studies

Male C57BL/6J mice (CLEA Japan, Inc.) were housed in accordance with our institutional guidelines and the Japanese Physiological Society’s guidelines for animal care in an air-conditioned room with a 12-h light/dark cycle, with food and water available ad libitum. Mice were divided into three groups and fed a standard chow diet (MF diet, ORIENTAL YEAS CO.,LTD), high fat/high sucrose (HFHS) diet (30% fat, 20% sucrose) or HFHS diet supplemented with EPA (5% wt/wt) for 24 weeks from 4 weeks of age. At the end of the study, mice were anesthetized by the intraperitoneal injection of pentobarbital (100 mg/kg) and then sacrificed. All experimental protocols for animal studies were approved by the institutional committee on animal care at Jichi Medical University.

Stromal vascular fraction isolation

The stromal vascular (SV) fraction was isolated from epididymal adipose tissue according to previously described methods with some modifications [11, 12]. In brief, after sacrifice, epididymal adipose tissues were resected, minced (<2 mm), and digested with collagenase (Sigma-Aldrich) in Krebs-Henseleit-HEPES buffer, pH 7.4, containing 20 mg/mL of BSA and 2.8 mM glucose at 37 °C using a shaker for 45 min. Then, samples were passed through a 40-μm mesh and centrifuged (1,000 g for 8 min). Pellets and floating cells were collected as the SV fraction and primary adipocytes, respectively. Isolated cells were then used for RNA extraction.

RNA was isolated from 3T3-L1 adipocytes and primary adipocytes with Direct-zol™ RNA MiniPrep (Zymo Research). Complementary DNA was made with ReverTra Ace qPCR RT Master Mix (TOYOBO). Reverse transcription polymerase chain reaction (RT-PCR) was performed using TaqMan® gene expression assays (Applied Biosystems). Primer-probe sets are shown in Additional file 1: Table S1. All mRNA levels were normalized by GAPDH, as an internal control gene. Relative gene expression levels were calculated using the ΔΔCT method as previously described [13]. Gene silencing of GPR120 was performed using mouse GPR120 siRNA (Santa cruz) according to the manufacturer’s protocol. The siRNA efficiency was determined using western blotting.

Histological analyses

Macrophage infiltration into epididymal adipose and hepatic tissues was evaluated using MAC-2 immunostaining. MAC-2 antibody staining and hematoxylin and eosin (HE) staining were performed at Biopathology Institute Co,. Ltd. (Oita, Japan). The number of crown-like structures (CLSs) was counted in five independent fields and recorded as the mean number of CLDs per low-power field (Olympus BX-51, ×100). Adipocyte diameter was measured as the mean diameter of 100 adipocytes in each slide.

Statistical analysis

All data are shown as means ± SEM. Statistical significance was determined using the unpaired Student's t-test. Spearman’s correlation was used for linear regression analysis. Prism version 5 (GraphPad software Inc.) was used for statistical analyses. P-values of <0.05 were considered statistically significant.

TLR4 signal transduction also accelerates complex formation between TAK1 and TAB1, a key signal transducer of NF-κB in macrophages, and DHA inhibits TAK1/TAB1 complex formation [6]. In the present study, we demonstrate EPA has anti-inflammatory activity by inhibiting TAK1/TAB1 complex formation in 3T3-L1 adipocytes; however, protein expression of TAB1 did not change following palmitate exposure. These results indicate TAK1/TAB1 complex formation is more important than TAB1 protein expression levels. Recently, a GPR120-specific agonist was reported to reduce the interaction between TAK1 and TAB1 in intestinal epithelial cells [21]. Taken together, these results indicate GPR120-mediated anti-inflammatory pathways may represent a common system in GPR120-expressed tissues. In cultured human adipocytes, silencing of GPR120 has been shown to abolish the suppressive effect of TNF-α gene expression in response to DHA [22]. However, the results of the present study are not inconsistent with those of previous studies.

In addition to the above-mentioned adipogenesis aspects, it has been found that white adipose tissue could change their phenotype by some stimulation [34]. UCP1 is a key protein to regulate energy expenditure of brown adipocytes and is also expressed in beige cells, which is induced from white adipose tissue [37]. It is known that beige cells are mitochondria-rich adipocytes and thermogenic activity, then could be inducible by cold, norepinephrine, irisin and fibroblast growth factor 21 stimulations [34]. Recently, EPA or fish oil induces UCP1 and changes the phenotype of white adipocytes to beige-like adipocytes [38, 39]. We found EPA supplementation increased UCP1 mRNA expression. EPA-induced beiging of adipocytes might contribute to the improvement of metabolic profile, however, whether these actions depends on GPR120 stimulation by EPA remains unclear. More detail study is needed to elucidate this point.

However, the potential effect of EPA on adiponectin protein or gene expression in vivo remains controversial. The above study reported the administration of omega-3-acid ethyl esters had no effect on adipocyte size or plasma adiponectin concentrations in a population of obese individuals [28]. These differences may be induced by EPA in vivo. In animal studies, dietary EPA doses are typically 5% (wt/wt) or more. However, the administration of doses at these levels in daily dietary forms is clinically challenging. Accordingly, comparisons between animal and human studies should be interpreted with caution, especially among nutrient supplementation studies.

Otherwise, improved insulin resistance may be an effect of decreased MCP-1 gene expression in adipose tissue, thereby reducing the involvement of M1 macrophage in adipose tissue. HFHS diet-induced adipose inflammation may therefore be improved. We demonstrated EPA supplementation reduces expression of the M1 macrophage marker, CD11c, in the SV fraction. In fact, several in vitro and in vivo studies have reported that EPA-treatment inhibits palmitate-induced MCP-1 induction in cultured adipocytes [14] and HFHS diet-induced MCP-1 induction in adipose tissues [24]. A direct effect of EPA on adipocytes may contribute to anti-inflammatory action via suppression of MCP-1 gene expression in adipose tissues. Oh et al. demonstrated that ω-3 fatty acid supplementation has no effect in macrophage specific GPR120 knock-down mice [6]. However, whether the effect of ATM polarity shift depends on macrophage or adipose GPR120 has yet to be elucidated.

In the present study, EPA inhibited HFHS diet-induced inflammation. Sato et al. reported similar results, with 20 weeks of EPA supplementation found to suppress BW gain and MCP-1 gene expression in HFHS diet-fed mice, but to have no effect in high-fat (HF) diet-fed mice [24].

Acknowledgments

Funding

This work was supported by a Grant-in-Aid for Scientific Research (C) (5001–89) from the Japan Society for the Promotion of Science (JSPS) to T.U., and JMU Graduate Student Start-Up Grant for Young Investigators to H.Y. This study was subsidized by JKA through its promotion funds from KEIRIN RACE to Jichi Medical University Saitama Medical Center.

Availability of data and materials

All datasets are included within this article.

Authors’ contributions

HY planned research design, collected the data, and wrote the manuscript. TU collected and analyzed data. MKakei, SM, MKawakami, SI, and KH analyzed data and edited the manuscript.

Competing interests

The authors declare that they have no competing interests

Consent for publication

All authors read and approved the final manuscript.

Ethics approval

This study was carried out in accordance with the Japanese Physiological Society’s guidelines for animal care. All experimental protocols for animal studies were approved by the institutional committee on animal care at Jichi Medical University.

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